This is the second year of operation of the Systems Neuroscience Imaging Resource (SNIR). Much of the focus has been on equipment acquisition and procedure development. A Zeiss AxioscanZ1 slide scanning microscope is now in its second year of operation. This is a high quality widefield microscope with transmitted brightfield and fluorescent epi-illumination capacity. Its most significant feature is the ability to program multichannel tiled acquisition of large areas from up to 100 microscope slides. It is being actively used by investigators from 10 different intramural laboratories for projects that include whole brain mapping of gene expression profiles and the projections of genetically tagged and fluorescently labeled neuron populations. To improve throughput the illumination system was upgraded this year to a 7 channel LED system. There have been technical issues with evenness of illumination. The system continues to be used to capacity. A Zeiss LSM780 microscope is approximately 5 years old. It is a high quality inverted confocal microscope with 405, 488, 514, 561, 594 and 633 nm lasers, a 32-channel GaAsP based spectral detector and 2 conventional PMTs. It is generally used to capacity during core working hours by investigators from approximately 10 different NIMH intramural groups. A LaVision Ultrascope was acquired and installed this year. This is a light sheet microscope optimized for low magnification (1.2 to 12X 0.5 NA objective with a light sheet thickness minimum of 4 microns) imaging of large samples (up to approximately 10 x 10 x 6 mm). It has 405, 488, 552, 638, and 740 nm lasers. Whole mouse brains immunolabeled with the iDISCO technique are being imaged and projects using other types of material are under development. A Leica SP8 confocal/multiphoton system was acquired this year and is largely set up. It is on an upright microscope frame equipped and with long working distance dipping objectives designed for work with thick cleared samples. It is equipped with 405, 488, 552 and 638 nm fixed lasers and an Insight X3 tunable IR laser and both internal and external non-descanned PMT and HyD detectors. A Nikon C2 system is being set up for both widefield epillumination and laser scanning confocal imaging. The microscope will provide alternatives to the Zeiss Axioscan and LSM780 that are being used to capacity. It will also provide the unique capacity to scan large areas in widefield mode at low or moderate magnification and to subsequently image regions of interest at high resolution and in confocal mode. Microbrightfield Brainmaker and Neurolucida 360 software packages are available to facilitate reconstruction and analysis of systems neuroscience data. A copy of Arivis Vision4D has been acquired and installed on a powerful PC workstation. Progress has been made on implementation of protocols for multiplex in situ hybridization on slabs of brain tissue up to 1mm thick.